Although various chemotherapeutic drugs have been found effective against certain tumors and even curative against some (Pardee, Devita and Hellman, eds., in Cancer, Principles and Practice of Oncology, Lippincott & Co. (1982)), there is a great need for therapeutic agents which kill cancer cells more efficiently and more selectively. An attractive approach towards meeting this need is to use antibodies to prepare antibody-drug complexes or "immunoconjugates" that direct or "target" anti-cancer agents to tumors. Antibodies are known in the art which recognize antigens expressed on cancer cells, for example the antibody 96.5 which reacts with the p97 antigen of human melanomas (Brown et al., J. Immunol., 127 p. 539 (1981)). Several immunoconjugates of this type have been shown to be selectively cytotoxic to antigen-positive tumor cells in vitro, to localize in tumors in vivo, and to have anti-tumor activity in mice that is greater than that of the drug or antibody alone (Rowland et al., Cancer Immunol. Immunotherapy, 19, pp. 1 (1985)). While the ability of such immunoconjugates to cure human tumors remains to be demonstrated, improvements in tumor targeting have been the focus of recent research efforts.
For a chemotherapeutic agent to be able to exert an effect on tumors, it must be taken up by the tumor cells, since very few, if any, cancer drugs are otherwise cytotoxic. The immunoconjugates must, therefore, be directed to the cancer cells, for example by antibody recognition of tumor-associated antigens, and either be taken up by the cancer cells (with active drug being released inside the cells), or the active drug must be released in the close vicinity of the cancer cells, and internalized in the same way as when the drug is used conventionally. The second alternative has several advantages. First, while anti-cancer drugs can be taken up by most cells, the internalization of immunoconjugates depends on both the antigenic target of the respective antibody and the cell in which the antigen is expressed. Antibodies to antigens that undergo modulation, i.e., those antibodies that are internalized in the form of an antigen-antibody complex (Old et al., Proc. Soc. Exp. Biol. Med., 124, p. 63 (1967)), are the ones most easily used for drug targeting (Jansen et al., Immunol. Rev., 62, p. 185 (1982)). Second, there is heterogeneity in the expression by cells of most tumor antigens so that cells which do not express a given antigen, i.e., are antigen-negative, frequently occur within a tumor (Yeh et al., J. Immunol., 126, p. 1312 (1981); Albino et al., J. Exp. Med., 154, p. 1764 (1981)). Although the difficulty of accumulating effective levels of chemotherapeutic agents within a tumor as a result of tumor cell heterogeneity can be decreased by combining antibodies to different antigens expressed by the same tumor cells and forming immunoconjugates, it could be further minimized if a therapeutic approach was developed in which the presence of some minimal amount of cells possessing the given antigen within a tumor would be sufficient to allow localization of effective amounts of immunoconjugates. Third, there are some tumor antigens, mucins, for example, which are present in larger amounts outside of the cells than at the cell membrane, (Rittenhouse et al., Laboratory Medicine, 16, p. 556 (1985)) suggesting the potential for targeting tumor regions.
The acidity (pH) of tumor tissues appears to be lower than that of normal tissues. Studies conducted more than half a century ago showed that malignant tumors metabolize carbohydrates mainly by anaerobic glycolysis, even under aerobic conditions (Warburg et al., Biochem. Z., 152, p. 309 (1924)). The oxidation of glucose stops at the stage of glucose oxidation to pyruvic acid, followed by reduction to lactic acid (Boxer and Devlin, Science, 134, p. 1495 (1961)). Most of this lactic acid is either removed or buffered by surrounding extracellular fluid, but some of it accumulates extracellularly. This results in a lower pH within the tumor than in normal tissues. Elevation of the blood-sugar by intravenous infusion of glucose should accelerate anaerobic metabolism resulting in even more lactic acid in the tumor, and this should further increase the pH difference between tumors and normal tissues.
Following Warburg's studies, there have been several reports of lower pH in tumors of both experimental animals, (Voegtlin et al., Nat'l. Inst. Hlth. Bull., 164, p. 1 (1935); Kahler and Robertson, J. Nat. Cancer Inst., 3, p. 495 (1943); and human patients, Naeslund, Acta Soc. Med. Upsal., 60, p. 150 (1955); Pampus, Acta Neurochir., 11, p. 305 (1963)).
Meyer et al., in Cancer Res., 8, p. 513 (1948) reported that the pH of malignant human tumors is lower than in normal tissues. In twelve out of fourteen cases, where both normal and neoplastic tissues from the same patients could be studied in vivo, there was a difference in pH which averaged 0.49 and ranged from 0.17 to 1.15.
Ashby, (Lancet, August 6, p. 312 (1966)), found that the mean pH of malignant tumors from nine patients was 6.8 (ranging between 6.6 and 6.9). Raising of the blood sugar by intraveneous infusion of dextrose further decreased the tumor pH to a mean of 6.5 (range 6.3-6.8).
Van Den Berg et al., Eur. J. Cancer Clin. Oncol., 18, p. 457 (1982), showed that the pH of twenty-two human mammary carcinomas was 7.29 (.+-.0.05, SEM), as compared to 7.63 (.+-.0.03, SEM) in human subcutis, and observed similar differences in rat tumors. The differences between pH in tumors and normal tissues were highly statistically significant, although they were lower than those reported in the studies discussed above.
Thistlethwaite et al., Int. J. Radiation Oncology Biol. Phys., 11, p. 1647 (1985), showed, likewise, that the pH of human tumors as measured by readings on fourteen tumors was below the physiological level with an average of 6.81.+-.0.09 (SEM). They speculated that the reported therapeutic effectiveness of hyperthermia depends on the lower extracellular pH of tumors as compared to normal tissues.
Trouet et al., U.S. Pat. No. 4,376,765, describe drug compounds composed of a protein macromolecule (carrier) linked via a peptide chain ("spacer arm") to an amino function of a drug. The carrier facilitates endocytic take-up by target cells so that the spacer arm may be cleaved within the cell. Recently, attention has been directed to developing antibody drug conjugates which release a drug within a tumor cell once the conjugate has crossed the cell membrane and encountered acidic pH (3.5-5.5) within the cell. U.S. Pat. No. 4,569,789 by Blattler et al., describes chemical formation of conjugates using crosslinking structures which can link amino-group substances such as chemotherapeutic drugs to the sulfhydryl portion of a compound such as an antibody reactive with tumor cell surface antigens capable of crossing the tumor cell membrane. One limitation of such a method of forming conjugates is that the antibody must contain a sulfhydryl group. This reduces the number of possible drug-antibody conjugates which may be formed using such procedures.
In spite of the published evidence that tumors have lower pH than normal tissues, and that acid-cleavable complexes may be formed between antibodies and drugs, this evidence has not yet resulted in the development of immunoconjugates which are composed of antibodies reactive with tumor associated antigens and chemotherapeutic agents, and which could be targeted to tumor tissues and are capable of selectively releasing the chemotherapeutic agents in the presence of the lower pH of cancer tissues for uptake by the tumor cells, but not at the pH of normal tissue.